LETTER
doi:10.1038/nature13047
A committed precursor to innate lymphoid cells Michael G. Constantinides1, Benjamin D. McDonald1, Philip A. Verhoef1 & Albert Bendelac1
1
a
CLP
B and T cells
NK T cells 3 1 and
iILC2
ILC2
2
GFP 0
0
94
69
80
YFP
b
NK cells
DX5+ cells
DX5– cells
ILC1
GFP 22
17
74
CD4+ LTi
CD4– LTi
NCR+ ILC3
13
60
68
YFP
c
GFP 4
YFP
d Per cent YFP+
100 75 50 25 0
BM Lu iIL ng C2 LP L ILC L PL 2 N IL C LP R + C2 L IL LP CD C3 L 4– C LT D4 i + IE LT L Li IL i ve C 1 Li r DX ve 5 – r Sp DX le 5 + en Sp N le K Sp en Sp le B le en en T N K T
Innate lymphoid cells (ILCs) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers1–9. Their diversity and similarities with previously characterized natural killer (NK) cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 solely on the basis of cytokine properties10, but their developmental pathways and lineage relationships remain elusive. Here we identify and characterize a novel subset of lymphoid precursors in mouse fetal liver and adult bone marrow that transiently express high amounts of PLZF, a transcription factor previously associated with NK T cell development11,12, by using lineage tracing and transfer studies. PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.11DX52 ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of ID2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages13. These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILCs, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes. To study the expression pattern of Zbtb16 encoding the transcription factor PLZF, which directs the developmental acquisition of the innate effector program of NK T cells11,12,14,15, we inserted a sequence coding for a fusion of enhanced green fluorescent protein (GFP) and Cre downstream of an IRES after the last exon of Zbtb16 (Extended Data Fig. 1a). As expected, GFP was selectively expressed in the NK T lineage, with early developmental stages 1 and 2 showing higher levels than mature stage 3 cells, but was not found in the bone marrow common lymphoid precursor (CLP), T cells or B cells of PLZFGFPcre1/2 mice (Fig. 1a). In PLZFGFPcre1/2 mice carrying the ROSA26-floxstopyellow fluorescent protein (YFP) fate-mapping allele, nearly all NK T cells expressed YFP, as expected, although approximately 35% of cells in all lymphoid and myeloid lineages were also labelled (Extended Data Fig. 1b, c and data not shown). Because haematopoietic stem cells (HSC) did not express GFP but were already labelled by YFP, this ‘background’ reflected some expression of PLZF before the HSC stage, probably in multipotent embryonic cells. Indeed, after transfer of fluorescenceactivated cell sorting (FACS)-sorted YFP-negative bone marrow cells into lethally irradiated recipients, 94% of NK T cells still expressed YFP, whereas donor-derived CLPs, B cells and T cells were unlabelled (Fig. 1a). Thus, the experiments shown in Fig. 1 were conducted with such bone marrow chimaeras, although all results were confirmed in non-chimaeric reporter mice. Intriguingly, several innate lymphoid lineages, which arise from CLP, were also specifically labelled by YFP despite absence of GFP expression. Thus, ILC2 in the lungs, intestinal lamina propria (LP), peritoneal cavity and mesenteric lymph nodes were all fate-mapped (Fig. 1a, d, Extended Data Fig. 1b and data not shown). Immature ILC2 in the bone marrow (iILC2) expressed very low amounts of GFP, but were already maximally labelled by YFP, suggesting expression of a higher level of PLZF at an earlier stage of their development (Fig. 1a, d). Group 1 innate lymphocyte subsets showed heterogeneous PLZF tracing:
Figure 1 | ILC lineage tracing in PLZFGFPcre reporter mice. a–c, Top rows, expression of GFP by indicated cell-types of PLZFGFPcre1/1 mice (filled grey) and wild type (WT; open). Bottom rows, YFP expression in radiation chimaeras reconstituted with YFP2Lin2Sca-11cKit1 (LSK) bone marrow cells from PLZFGFPcre1/2 ROSA26-YFP mice. CLP and iILC2 from bone marrow (BM); B and T cells and NK cells from spleen; NK T cells from thymus (GFP), divided into early stages 1, 2 and late stage 3, and from spleen (YFP); ILC2 from lung; DX51 and DX52 NK cells from liver; ILC1 from intestinal intraepithelial lymphocytes (IEL); LTi and ILC3 from LPL. BM iILC2 and lung ILC2 were identified as Lin2CD251IL-7Ra1Thy1.21; LPL ILC2 as CD3e2CD192 CD251KLRG11; LPL NCR1 ILC3 as CD3e2CD192NKp461NK1.12; LPL CD42 LTi cells as CD3e2CD192IL-7Ra1KLRG12CCR61CD42; LPL CD41 LTi cells as CD3e2CD192CCR61CD41; and IEL ILC1 as CD3e2CD192 NKp461NK1.11CD1601. FACS identification of the remaining subsets is defined in the methods section. d, Summary of results (mean 6 s.e.m.). Data representative of 3–9 individual mice analysed in at least 2 independent experiments.
Committee on Immunology, Department of Pathology, The Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA. 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 1
©2014 Macmillan Publishers Limited. All rights reserved
RESEARCH LETTER whereas few splenic NK cells expressed YFP, intestinal intraepithelial NK-like cells (termed ILC11) were prominently labelled (Fig. 1b). In the liver the recently described non-recirculating DX52CD49a1 subset of CD3e2NK1.11 cells16 was heavily labelled, whereas classical recirculating DX51CD49a2 NK cells were mostly negative. Different subsets of group 3 innate lymphocytes in the LP also showed markedly different patterns of tracing (Fig. 1c and Extended Data Fig. 2). CD41 and CD42 LTi were not labelled, whereas NCR1 ILC3 prominently expressed YFP. In summary, PLZF lineage-tracing labelled not only ILC2 but also the subsets of group 1 and group 3 cells that are most clearly distinguishable from classical NK and LTi cells, respectively, and will hereafter be termed ILC1 and ILC3. Searching for the PLZF-expressing precursor of ILCs, we identified a rare subset of PLZFhigh cells in fetal liver and adult bone marrow. They showed a homogeneous lineage2(Lin2)IL-7Ra1cKit1a4b7high phenotype (Fig. 2a, b), similar to the CLP-derived subset previously suggested to contain precursors for LTi17–20. The PLZFhigh population represented approximately 5% of Lin2IL-7Ra1cKit1 cells and approximately 30% of the a4b7high fraction (Fig. 2b, c). It expressed Thy1 but lacked expression of markers associated with mature ILCs, NK or LTis such as CD4, CXCR6, CD25, IL-17RB, T1/ST2, Sca-1, CD122, NK1.1, CCR6 and NKp46 (Fig. 2c, d and data not shown). Interestingly, the PLZFhigh cells included a fraction of CD62LhighICOSlow cells, probably representing the earliest developmental stage after PLZF expression, because PLZF characteristically induces the downregulation of CD62L and upregulation of ICOS15,21. Transcriptional analysis of purified a
b Lin– IL-7Rα+ cKit+ E14 fetal liver d
Adult bone marrow PLZFGFPcre+/+
WT
PLZFGFPcre+/–
WT 12
8
0
CD25
CD44
CD62L
ICOS
IL-17RB
Sca-1
T1/ST2
Thy1.2
4
0.01
α4β7
Lineage
0
PLZFhigh cells compared with bone marrow CLP, iILC2 and NK progenitors (NKP), and with ILC3-enriched lamina propria lymphocytes (LPL), confirmed the very high amounts of Zbtb16 mRNA encoding PLZF, as well as of other key transcription factors such as Id2, Gata3 and Rora, which are required for ILC2 development3,22–25 (Fig. 2e and Extended Data Fig. 3) and Tcf7, a target of Notch required for ILC2 and ILC326,27. Notably, they expressed very high amounts of Tox, which is required for the development of both NK and LTi cells13, and low but detectable amounts of Tbx21 and Rorc, the transcription factors associated with ILC1 and ILC3, respectively1,6. Single-cell analysis by intracellular flow cytometry was performed after MACS-depletion of Lin1 cells in fetal liver and adult bone marrow, gating on Lin2IL-7Ra1 a4b7high cells (Fig. 2f). PLZFhigh cells, which represented up to 40% of this population, characteristically co-expressed high amounts of both GATA3 and TOX. Interestingly, a small fraction of these PLZFhigh cells also expressed RORct, although these were mostly found in the fetal liver and were rare in adult bone marrow. A distinct population consisting of RORcthigh PLZF2 cells, which was more abundant in the fetal liver, co-expressed high levels of TOX but not GATA3, probably representing LTi precursors. Finally, a fraction of Lin2IL-7Ra1a4b7high cells lacked both PLZF and RORct, but expressed TOX. These may be earlier undifferentiated precursors to the ILC and LTi lineages, and may also include precursors to classical NK cells. To determine precursor-product relationships, we injected a 1:1 mixture of PLZFhigh cells and CLP cells into Rag22/2Il2rg2/2 mice. The PLZFhigh cells, which were purified from a ROSA-floxstop-tdTomato 3
GFP Lin– IL-7Rα+ cKit+ adult BM 0
0.01
PLZFGFPcre+/+
α4β7
WT
0
6
0
0.01
IL-7Rα
GFP Lin– IL-7Rα+ cKit+ α4β7high
c
E14 fetal liver 0.01
cKit
CXCR6
0
Adult BM
18
1
24
0
58
23
42
32
GFP
C LP
2
gh
C
F hi
Adult BM
40
12
7
1
18
30
49
43
10
33
18
46
Tcf7
Tox 6
47
10
31
5
44
38
35
44
10
8
21
0
4 TOX
2 2
KP
PLZF
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C
gh
C LP PL ZF hi
2 C
hi g
iIL
h
0 C LP PL ZF
2 C iIL
gh
F hi
PL Z
C LP
8 6 4 2 0
iIL
Id2
5 4 3 2 1 0
GATA3
IL
C
PL
iIL
C LP
PL Z
gh hi
iIL C 2
ZF
PL
C LP
0
RORγt
10
E14 fetal liver
gh
20
hi
30
Lin– IL-7Rα+ α4β7high
f
Rorc
2.5 2.0 1.5 1.0 0.5 0.0
C 32 e LP nric L he d
Gata3
Zbtb16 5 4 3 2 1 0
ZF
e
iIL
GFP
Expression relative to Hprt1
5
α4β7
12
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©2014 Macmillan Publishers Limited. All rights reserved
Figure 2 | PLZFhigh cells in fetal liver and bone marrow. a, FACS analysis of total BM cells from adult WT littermate and PLZFGFPcre1/1 mice. Data representative of at least 3 independent experiments. b, c, GFP expression is confined to the a4b7high population of indicated fractions in fetus and adult. Representative of at least 3 independent experiments. d, FACS analysis of PLZFhigh BM cells (solid line) and BM iILC2 (dashed line) compared with isotype controls (filled). iILC2 were gated as Lin2ICOS1T1/ST21Sca-11Thy1.21 for CD25 profile, as CD251CD62L2Sca-11Thy1.21 for CD44 profile, as CD251CD441Sca-11Thy1.21 for CD62L profile, as CD251T1/ST21Sca11Thy1.21 for ICOS profile, as CD251ICOS1Sca11Thy1.21 for T1/ST2 profile, as CD251Thy1.21 for Sca-1 profile or as CD251ICOS1Sca-11 for Thy1.2 profile. Representative of at least 3 independent experiments. e, Quantitative PCR with reverse transcription analysis in bone marrow subsets as indicated. NKP are Lin2CD271IL7Ra2Flt32CD1221 BM cells and ILC3-enriched LPL are CD3e2CD192IL7Ra1Thy1.21KLRG12CD42 cells. Mean 6 s.e.m. of data from 2–3 independent experiments. f, Intracellular FACS analysis for transcription factors in cells as indicated. Representative of at least 3 independent experiments.
LETTER RESEARCH PLZFGFPcre1/2 background to further ascertain their history of PLZF expression based on tdTomato expression, gave rise to a diversity of innate lineages, with the notable exception of CD41 LTi (Fig. 3a). Significantly more peripheral ILC2 were derived from the PLZFhigh b
CD45.1
CD19
4
PLZFhigh
97
CD3ε
Liver
All lymphocytes 22
28
Lineage
*
*** ***
15
75 50 25
e
Days in culture
d
1
CD25
*** ***
LP
DX5
All lymphocytes 2 22
Lung
***
L
CD25
57
**
100
0
NK1.1+ CD3ε– TCRβ– 78
CD49a
2
c
All lymphocytes
tdTomato
CLP
PLZFhigh
CLP
1.3
BM Lu iILC ng 2 RO IL Rγ L t + PL C2 LP NC IL LP L R + C2 R L RO OR (ILC Rγ γt + 3) t+ N C CR D4 – + ( IE LTi) L Li ILC ve Lu r 1 L DX n Sp g N ive 5 – le K1 r D en .1 + X5 + N K1 CD .1 + 3ε – C D Sp 3ε – le e Sp n B le en T
6.8
Per cent PLZFhigh-derived
a
fraction at 5–7 weeks post-injection (Fig. 3a, c). Both progenitors gave rise to similar amounts of CD3e2NK1.11 group 1 cells, but CLP mostly generated the classical DX51CD49a2NK1.11 NK cells, whereas the PLZFhigh cells primarily generated a distinct DX5–CD49a1NK1.11 ILC1
Post-sort
OP9
OP9-DL1
95
1
53
5
54
34
4 GFP
0
40
1
12
1
2
3
4
11
9
35
14
33
6
35
2
28 GFP
52
22
29
54
7
61
2
15
5
35
14
33
6
33
4
62
3
48
20
41
8
55
α4β7
All lymphocytes 73
NK1.1
3
2B4
f
Adult bone marrow
All lymphocytes 52 43
CD19
Spleen
Fetal liver
PLZFhigh 43 6 42
CLP
0 0
ICOS
IL-7Rα
PLZFhigh 22 30 30
g
PLZFhigh single cells on OP9 ILC1,2
TCRβ
56
0
0
19
33
ILC1,2,3
0
35
35
64
32
30
64 α4β7
3
54
11
1
0
35
2
29
4
35
0
99
0
60
3
32
23
42
1
0
2
35
45
RORγt
94 IL-17RB
6
2
0
0
11
41
18
42
6
46
α4β7
Sca-1
CD3ε– CD19– lymphocytes 13 47
KLRG1
82 CD25
ICOS
0
high
18 24
44
49 CD19
ICOS
58
18
35 NK1.1
28
ICOS
33
0
ILC2,3
6
RORγt 0
52
0
51
48
0
20 NKp46
CD4
8
ILC1,3
28 NK1.1
NK1.1
CD3ε– CD19– lymphocytes
LPL
18 CD122
RORγt 2/2
2/2
Figure 3 | PLZF cells are ILC progenitors. a–c, CD45.2 Rag2 Il2rg mice were injected with equivalent numbers of tdTomato1 PLZFhigh cells and CD45.1 CLP (800–1,200 of each) and the progeny of these populations analysed 5–7 weeks later by FACS, as indicated. Summary bar graph of mean percentages 6 s.e.m. in c, with significant differences between PLZFhigh- (black bar) and CLP-derived (white bar) shown by *. LPL RORct1 NCR1 cells were identified as CD3e2CD192RORct1NKp461CD42; LPL RORct1NCR2 cells as CD3e2CD192RORct1NKp462CD42; and LPL RORct1CD41 cells as CD3e2CD192RORct1CD41. Remaining populations were gated as indicated in Fig. 1. Data representative of 6–9 chimaeras analysed in at least 2 independent experiments. d, FACS analysis of Lin2IL7Ra1a4b7highGFP2CXCR62 cells sorted from PLZFGFPcre1/2 BM and cultured on OP9 or OP9-DL1 for 48 h. Data representative of 3 independent experiments. e, FACS analysis of sorted PLZFhigh BM cells cultured on OP9 for indicated periods. Data representative of at least 4 replicate cultures for each time point from 2 or more independent experiments. f, FACS analysis of
ILC Number of colonies type Exp 1 Exp 2 Exp 3 Exp 4 1 1,2,3 2 0 4 0 1,2 3 2 4 2 1,3 6 3 10 10 2,3 10 0 1 45 ILC1 50 35 100 42 ILC2 30 16 53 ILC3 3 11 35 22
Total 7 9 21 21 230 141 71
Colony size 200 ± 32 156 ± 32 134 ± 23 96 ± 15 72 ± 8 33 ± 2 49 ± 7
PLZFhigh or CLP cells from adult BM or fetal liver cultured on OP9 for 4 days. Data representative of at least 4 replicate cultures from 2 or more independent experiments. g, FACS analysis of representative colonies originating from single fetal liver PLZFhigh cells that were sorted and cultured into 96-well plates containing irradiated OP9 stromal cells for 5–6 days. ILC1 were characterized as ICOSlowa4b72 NK1.11 populations, ILC2 as ICOShigha4b72NK1.12, and ILC3 as ICOSint/higha4b71NK1.12. The table summarizes the analysis of thirteen 96-well plates analysed in four independent experiments (three plates in experiments 1, 2 and 4; four plates in experiment 3), with the average colony size 6 s.e.m. as indicated. In experiment 4, we mixed a 1:1 ratio of CD45.1/5.2 and CD45.2 PLZFGFPcre1/2 fetal liver cells before single-cell sorting. All 122 colonies were either CD45.1/5.2 (n 5 59) or CD45.2 (n 5 63), ruling out doublet contamination as an explanation for the presence of mixed ILC colonies (P , 0.01 Chi-square). The cloning efficiency was 40% on average, with all but 17 colonies (not shown in the table) unambiguously assigned to defined ILC lineages. 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 3
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RESEARCH LETTER b
12 77
800
13
CD45.1 1.5
600 400 200
ICOS ***
100 80 60 40 20
–/
+
–
0
+/
KO:WT ratio
0
1.0
120
–
CD45.2
19
CD4+ LTi
–/
ILC2
ICOS
CD62L *
+/ +
LPL
CD62L RORγt
Per cent of WT MFI
KLRG1
c
6
CD4
CD25
CD3ε– CD19– lymphocytes 3
Per cent of WT MFI
a
** 0.5
*** ***
***
0.0 BM i LP Lu ILC L ng 2 RO IL Rγ C L t + PL 2 N LP C ILC R+ 2 LP L R ( L RO OR ILC Rγ γt + 3) t + NC C D4 R – + (L IE Ti) L I L Li ve C1 r Li DX5 ve – rD X5 +
subset in the liver (Fig. 3b, c). PLZFhigh-derived NK1.11CD3e–TCRb– cells were observed in the spleen and lungs, and expressed significantly more NKp46 than CLP-derived NK cells (Extended Data Fig. 4). Unlike CLP, PLZFhigh cells did not generate B cells, consistent with their expression of Tcf7, suggesting that they had already received a Notch signal in vivo. They also gave rise to far fewer T cells than CLP after transfer into Rag22/2Il2rg2/2 mice in vivo (Fig. 3a, c) or after coculture with fetal thymic stroma in vitro (Extended Data Fig. 5), indicating that PLZFhigh cells had also largely lost T cell potential. Thus, the PLZFhigh cell is an ILC precursor (ILCP) heavily committed to innate lymphoid lineages, excluding classical NK and LTi cells. When cultured on OP9 stromal cells, the GFP2 fraction of Lin2IL7Ra1cKit1a4b7high cells generated a small fraction of GFP1 cells within 2 days of culture, which was increased upon culture on OP9DL1 cells (Fig. 3d), confirming the precursor–product relationship and its promotion by Notch signals. To study the short-term fate of ILCP in vitro, we sorted Lin2IL-7Ra1cKit1a4b7high PLZFhigh cells from either adult bone marrow or fetal liver and cultured them on OP9 stromal cells, without Notch ligands, in the presence of non-polarizing IL-7 and stem-cell factor (SCF). By 24 h, a substantial fraction of the PLZFhigh population had already begun to downregulate GFP and to upregulate either ICOS or CD122 (Fig. 3e), and by 4 days, the cells had resolved into three separate populations of ILC1 (ICOS2IL-17RB2T1/ ST22CD252RORct2GATA32a4b72CD1221NK1.11NKp461), ILC2 (ICOShighIL-17RB1T1/ST21CD251RORct2GATA31a4b72CD1222 NK1.12NKp462), and ILC3 (ICOSintIL17RB2T1/ST22CD252RORct1 GATA32a4b71CD1222NK1.12NKp462/low) (Fig. 3f and Extended Data Fig. 6). This transient expression of PLZF before ILC differentiation may explain why fate-mapping by the ROSA-YFP allele in Fig. 1 did not quite reach 100% for these populations as in NK T cells where PLZF expression is permanent. Notably, ILC3 cells consistently arose with higher frequency from fetal liver than from adult bone marrow cells (Fig. 3f). In single-cell cultures with OP9 stromal cells, fetal PLZFhigh cells generated mixtures of ILC lineages, including triple and double ILC1, ILC2 and ILC3, in 58 out of 500 wells (12%) as well as single ILC1, ILC2 or ILC3 populations in the remaining wells (Fig. 3g). Mixing CD45 allele-marked fetal liver cells before single-cell sorting confirmed that the mixed ILC colonies did not result from cell aggregates escaping exclusion during cell-sorting (Fig. 3g, experiment 4) and post-sorting microscopic assessment confirmed single-cell seeding in each of 96 wells. These results indicated that the PLZFhigh cells contained a common ILC progenitor characterized by multiple ILC lineage potential at the clonal level, and that subsequent commitment to individual ILC1, ILC2 or ILC3 lineages occurred rapidly during the PLZFhigh stage of ILC development. Wells containing mixtures of ILC lineages had more growth than those with single lineages, indicating that multipotent ILC progenitors retained greater proliferative capacity. We then assessed the contribution of PLZF to ILC development in lethally irradiated recipients of a mixture of bone marrow from congenically labelled Zbtb162/2 and Zbtb161/1 littermate donors (Fig. 4a). The production of ILC2 was markedly decreased in the PLZF-deficient compartment of all tissues, demonstrating a cell-intrinsic requirement of PLZF. Furthermore, residual immature ILC2 in the bone marrow expressed significantly less ICOS and more CD62L in the absence of PLZF (Fig. 4b, c), a phenotype entirely consistent with that of PLZFdeficient NK T cells11,12,15, which accounts for altered recirculating properties and reduced frequencies in peripheral tissues. LTi cells were unaffected by the genetic deletion of PLZF, as expected (Fig. 4a). Other ILC3 cells, including NCR1 ILC3, were unaffected, despite their expression of PLZF during development. However, liver ILC1 (DX52CD49a1) were profoundly affected, similar to ILC2, whereas classical (DX51CD49a2) NK cells were not (Fig. 4a). Our studies have identified the elusive CLP-derived common progenitor to the ILC1, ILC2, and ILC3 lineages and demonstrated close but distinct lineage relationships with classical NK and LTi cells. We have shown that a committed PLZFhigh ILCP emerges from within the
Figure 4 | PLZF is required for ILC development. Lethally irradiated CD45.1/2 mice were reconstituted with a mixture of CD45.2 Zbtb162/2 and CD45.1 WT BM and analysed as indicated 5–7 weeks later. a, Representative FACS analysis of LPL gated as indicated, and summary bar graph of mean percentages 6 s.e.m. for indicated populations, with significant variation from 1.00 indicated by *. Populations were gated as indicated in Fig. 3. Data representative of 6–10 chimaeras analysed in at least 2 independent experiments. b, c, FACS analysis of iILC2 from Zbtb162/2 mice and Zbtb161/1 littermates, representative of 5 (b) and 8 (c) mice of each genotype analysed in 3 or more independent experiments.
same IL-7Ra1a4b7high population that includes precursors to LTi and possibly also to NK cells (Extended Data Fig. 7). It is uniquely distinguished and affected by the rapid and transient upregulation of PLZF, whereas TOX is more broadly induced and reciprocally controls classical NK and LTi cells. The concomitant induction of GATA3, RORa, Tbet and RORct, which govern the polarization of ILCs, parallels the sequence during PLZF-dependent NK T cell differentiation28, indicating a broad and defining role of PLZF in the differentiation of innate lymphocyte lineages.
METHODS SUMMARY PLZFGFPcre mice were generated by inserting a sequence encoding an IRES and EGFP–Cre fusion protein immediately after the Zbtb16 stop codon through homologous recombination in C57BL/6J embryonic stem cells. For fate-mapping, they were crossed to mice carrying a ROSA26-fl-STOP-fl-YFP or a ROSA26-fl-STOPfl-tdTomato allele. Bone marrow chimaeras were generated by reconstituting lethally irradiated recipients with purified Lin2 Sca-11 cKit1 (LSK) bone marrow cells. PLZFhigh ILC precursors were purified from bone marrow or fetal liver and cultured in bulk or as single cells deposited by flow cytometry in 96-well plates with irradiated OP9 or OP9-DL1 stromal cells and murine IL-7 and SCF. For in vivo transfers, PLZFhigh ILC precursors and CLP were purified from bone marrow and injected into Rag22/2IL2rg2/2 mice. Quantitative PCR with reverse transcription for transcription factors was performed using TaqMan primers. Online Content Any additional Methods, Extended Data display items and Source Data are available in the online version of the paper; references unique to these sections appear only in the online paper. Received 27 November 2013; accepted 21 January 2014. Published online 9 February 2014. 1. 2. 3.
Fuchs, A. et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL12- and IL-15-responsive IFN-c-producing cells. Immunity 38, 769–781 (2013). Neill, D. R. et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464, 1367–1370 (2010). Moro, K. et al. Innate production of TH2 cytokines by adipose tissue-associated c-Kit1Sca-11 lymphoid cells. Nature 463, 540–544 (2010).
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LETTER RESEARCH 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
18.
Price, A. E. et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proc. Natl Acad. Sci. USA 107, 11489–11494 (2010). Monticelli, L. A. et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nature Immunol. 12, 1045–1054 (2011). Satoh-Takayama, N. et al. Microbial flora drives interleukin 22 production in intestinal NKp461 cells that provide innate mucosal immune defense. Immunity 29, 958–970 (2008). Sanos, S. L. et al. RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp461 cells. Nature Immunol. 10, 83–91 (2009). Luci, C. et al. Influence of the transcription factor RORct on the development of NKp461 cell populations in gut and skin. Nature Immunol. 10, 75–82 (2009). Cella, M. et al. A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature 457, 722–725 (2009). Spits, H. et al. Innate lymphoid cells–a proposal for uniform nomenclature. Nature Rev. Immunol. 13, 145–149 (2013). Savage, A. K. et al. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 29, 391–403 (2008). Kovalovsky, D. et al. The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nature Immunol. 9, 1055–1064 (2008). Aliahmad, P., de la Torre, B. & Kaye, J. Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages. Nature Immunol. 11, 945–952 (2010). Savage, A. K., Constantinides, M. G. & Bendelac, A. Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling. J. Immunol. 186, 5801–5806 (2011). Constantinides, M. G., Picard, D., Savage, A. K. & Bendelac, A. A naive-like population of human CD1d-restricted T cells expressing intermediate levels of promyelocytic leukemia zinc finger. J. Immunol. 187, 309–315 (2011). Peng, H. et al. Liver-resident NK cells confer adaptive immunity in skin-contact inflammation. J. Clin. Invest. 123, 1444–1456 (2013). Yoshida, H. et al. Expression of a4b7 integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells. J. Immunol. 167, 2511–2521 (2001). Sawa, S. et al. Lineage relationship analysis of RORct1 innate lymphoid cells. Science 330, 665–669 (2010).
19. Possot, C. et al. Notch signaling is necessary for adult, but not fetal, development of RORct1 innate lymphoid cells. Nature Immunol. 12, 949–958 (2011). 20. Cherrier, M., Sawa, S. & Eberl, G. Notch, Id2, and RORct sequentially orchestrate the fetal development of lymphoid tissue inducer cells. J. Exp. Med. 209, 729–740 (2012). 21. Gleimer, M., von Boehmer, H. & Kreslavsky, T. PLZF controls the expression of a limited number of genes essential for NKT cell function. Front. Immunol. 3, 374 (2012). 22. Hoyler, T. et al. The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells. Immunity 37, 634–648 (2012). 23. Mjo¨sberg, J. et al. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. Immunity 37, 649–659 (2012). 24. Wong, S. H. et al. Transcription factor RORa is critical for nuocyte development. Nature Immunol. 13, 229–236 (2012). 25. Halim, T. Y. et al. Retinoic-acid-receptor-related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation. Immunity 37, 463–474 (2012). 26. Yang, Q. et al. T cell factor 1 is required for group 2 innate lymphoid cell generation. Immunity 38, 694–704 (2013). 27. Mielke, L. A. et al. TCF-1 controls ILC2 and NKp461RORct1 innate lymphocyte differentiation and protection in intestinal inflammation. J. Immunol. 191, 4383–4391 (2013). 28. Constantinides, M. G. & Bendelac, A. Transcriptional regulation of the NKT cell lineage. Curr. Opin. Immunol. 25, 161–167 (2013). Acknowledgements We thank W. Yokoyama for discussion; F. Gounari and R. de Pooter for advice on OP9 cultures; H. Gudjonson for statistical advice; D. Leclerc, J. Cao, M. Olsen and R. Duggan for help with cell sorting; V. Bindokas and R. Mathew for help with fluorescence microscopy. This work was supported by NIH grants R01HL118092, R01AI038339 and P30DK42086 and by The Howard Hughes Medical Institute (A.B.). Author Contributions M.G.C., B.D.M. and P.A.V. designed research, performed experiments and analysed data. M.G.C. and A.B. wrote the paper. A.B. supervised the research. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to A.B. ([email protected]).
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RESEARCH LETTER METHODS a
b
Mice. C57BL/6J (stock no. 000664), B6.SJL-Ptprc Pepc /BoyJ (CD45.1; stock no. 002014), B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (stock no. 006148), B6.CgGt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (stock no. 007914), and B6.Cg-Tg(ACTFLPe) 9205Dym/J (stock no. 005703) mice were obtained from The Jackson Laboratory, whereas B6.Rag2tm1Fwa II2rgtm1Wjl (stock no. 4111) mice were obtained from Taconic. Plzf2/2 mice were a gift from P. P. Pandolfi and were backcrossed to C57BL/6J for at least nine generations. Animals were 4–10 weeks of age when analysed and were compared to littermate controls. For fetal experiments, the morning a vaginal plug was observed was considered embryonic day 0 (E0). Mice were housed in a specific-pathogen-free environment at the University of Chicago and experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. Generation of the PLZFGFPcre reporter strain. The PLZFGFPcre strain was generated as follows. A sequence encoding an IRES and GFP–Cre fusion protein was cloned from the pIGCN21 plasmid29 and inserted immediately following the Zbtb16 stop codon. The linearized construct was transfected into C57BL/6J embryonic stem cells and neomycin-resistant clones were screened by PCR. Clones that had undergone homologous recombination were injected into albino C57BL/6J blastocysts and the resulting chimaeric mice were crossed with ACTB-FLPe mice to excise the neomycin resistance cassette. The following PCR primers were used to identify WT (296 base pairs) and knock-in (419 base pairs) alleles: 59-GTGTACC ATCTGCACGGAAT-39, 59-GGCCAAGTAACTATCAGGCT-39, and 59-TACC GGAGATCATGCAAGCT-39. Preparation of cell suspensions. Spleen, liver and lymph nodes were mechanically dissociated through 70-mm filters and bone marrow was isolated by gently crushing femurs and tibias before filtration. Following dissociation, each liver was centrifuged at 400g for 5 min, resuspended in 5 ml of 40% Percoll (Sigma-Aldrich), and then centrifuged at 800g for 10 min. The supernatant was aspirated and the cell pellet was resuspended in HBSS (Gibco) containing 0.25% BSA (Sigma-Aldrich) and 0.65 mg l21 sodium azide (Sigma-Aldrich). For the isolation of lung lymphocytes, mice were anaesthetized with ketamine/ xylazine and approximately 1 ml of PBS (Sigma-Aldrich) was injected into the right ventricle to perfuse the lung tissue. Pairs of lungs were diced and incubated in 5 ml of pre-warmed RPMI 1640 (Cellgro) containing 0.01% DNase I (Roche), and 650 units per ml collagenase I (Worthington) in a 37 uC shaking incubator for 30 min. The digested tissue was passed through a 70-mm filter and centrifuged at 400g for 5 min. The cells were resuspended in 5 ml of 40% Percoll, underlaid with 5 ml of 60% Percoll, and centrifuged at 800g for 20 min with no brake. Lymphocytes were isolated from the interface and resuspended in HBSS containing 0.25% BSA and 0.65 mg l21 sodium azide. To isolate IEL and LPL, the small intestine was emptied of its contents, opened by making a lengthwise incision, and cut into 1-cm pieces which were then shaken at 37 uC for 30 min in RPMI 1640 containing 1% FCS (Biowest), 1 mM EDTA (Fluka), and 1 mM DTT (Sigma-Aldrich). Following the incubation, the supernatant was centrifuged at 400g for 5 min, resuspended in 5 ml of 40% Percoll, and then centrifuged at 2,000g for 10 min, yielding the IEL fraction. The pieces of tissue were subjected to two 30-min digestions in RPMI 1640 containing 20% FCS, 0.2 mg ml21 DNase I, and 100 units ml21 collagenase VIII (Sigma-Aldrich). The digested tissue was passed through a 70-mm filter, centrifuged at 400g for 5 min, resuspended in 5 ml of 40% Percoll, and then centrifuged at 2,000g for 10 min to isolate the LPL fraction. Flow cytometry. Cell suspensions were incubated with purified anti-CD16/32 (clone 93) for 10 min on ice to block Fc receptors. Fluorochrome- or biotin-labelled monoclonal antibodies (clones denoted in parenthesis) against 2B4 (2B4), a4b7 (DATK32), B220 (RA3-6B2), CCR6 (29-2L17), CD3 (17A2), CD3e (145-2C11), CD4 (RM4-5 or GK1.5), CD8a (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD25 (PC61), CD27 (LG.7F9), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD49a (HMa1), CD62L (MEL-14), CD122 (5H4 or TM-b1), CD160 (7H1), cKit (2B8), CXCR6 (221002), DX5 (DX5), Flt3 (A2F10), Gr-1 (RB6-8C5), I-A/I-E (M5/ 114.15.2), ICOS (C398.4A), IL-17RB (752101), IL-7Ra (A7R34), KLRG1 (2F1), NK1.1 (PK136), NKp46 (29A1.4), Sca-1 (D7), T1/ST2 (D1H9), TCRb (H57-597), Ter-119 (TER-119), and Thy1.2 (53-2.1) were purchased from BD Biosciences, BioLegend, eBioscience or R&D Systems. CD1d-PBS57 tetramer was from the NIH tetramer facility. For pre-enrichment of fetal liver and BM PLZFhigh cells, samples were stained with allophycocyanin (APC)-conjugated anti-a4b7 antibody, bound to anti-APC microbeads (Miltenyi Biotec), and subjected to doublecolumn enrichment on an autoMACS (Miltenyi Biotec). For the isolation of HSC, CLP, BM iILC2 and lung ILC2, lineage1 cells were depleted by staining with biotin-conjugated antibodies against B220, CD3e, CD4, CD8a, CD11b, CD11c, CD19, Gr-1, NK1.1, TCRb and Ter119, followed by an incubation with SAv microbeads (Miltenyi Biotec). Fetal liver progenitors were similarly enriched, except the biotin-conjugated lineage antibodies used were against B220, CD3e, CD11c, CD19,
NK1.1, NKp46 and Ter119. To exclude dead cells, 49,6-diamidino-2-phenylindole (DAPI; Molecular Probes) was added to all live samples. Cells were run on an LSRII (BD Biosciences) or sorted using a FACS Aria II (BD Biosciences) and analysed using FlowJo software (Tree Star). Collected events were gated on DAPI2 lymphocytes and doublets were excluded. For the intracellular staining of fetal liver progenitors, sorted Ly5.2 Lin2IL7Ra1a4b71 cells were combined with 2 3 105 Ly5.1 thymocytes to increase the size of the cell pellet. Samples were stained with anti-CD45.1 and anti-CD45.2 antibodies and fixed and permeabilized using the Foxp3 staining kit (eBioscience). Samples were then stained with monoclonal antibodies against GATA3 (TWAJ), RORct (B2D), and/or Tox (TXRX10). Intracellular PLZF was detected by conjugating a purified monoclonal antibody (D9; Santa Cruz) to Alexa Fluor 647 using an antibody labelling kit (Molecular Probes). Unless otherwise indicated, PLZFhigh cells were identified and sorted as Lin2IL7Ra1a4b71EGFP1; CLP as Lin2IL-7Ra1cKitintSca-1intFlt3high; HSC as Lin2 cKit1Sca-11Flt32; BM iILC2 and lung ILC2 as Lin2CD251IL-7Ra1Thy1.21; LPL ILC2 as CD3e2CD192CD251KLRG11; LPL NCR1 ILC3 as CD3e2CD192 NKp461NK1.12; LPL RORct1 NCR1 cells as CD3e2CD192RORct1NKp461 CD42; LPL RORct1 NCR2 cells as CD3e2CD192RORct1NKp462CD42; LPL CD41 LTi cells as CD3e2CD192CCR61CD41; LPL RORct1 CD41 cells as CD3e2CD192RORct1CD41; LPL CD42 LTi cells as CD3e2CD192IL7Ra1KLRG12CCR61CD42; IEL ILC1 as CD3e2CD192NKp461NK1.11CD1601; liver DX51 cells as CD3e2TCRb2NK1.11DX51CD49a2; liver DX52 cells as CD3e2TCRb2NK1.11DX52CD49a1; spleen NK cells as CD3e2NK1.11; spleen DC as CD11c1I-A/I-E1; spleen T cells as TCRb1; spleen B cells as CD191; NK T cells as CD1d-PBS57 tetramer1 CD242 in the thymus and CD1d-PBS57 tetramer1 TCRb1 in the spleen. Lineage antibodies against the following markers were used: B220, CD3e, CD4, CD8a, CD11b, CD11c, CD19, Gr-1, NK1.1, TCRb and Ter119. OP9/OP9-DL1 cultures. Stocks of OP9 and OP9-DL1 stromal cells were a gift from J. C. Zu´n˜iga-Pflu¨cker. All experiments were performed in Opti-MEM with GlutaMAX (Gibco) containing 10% FCS (Gibco), 13 penicillin/streptomycin (Gibco), and 60 mM 2-mercaptoethanol (Sigma-Aldrich) and maintained in a 37 uC incubator (Thermo Scientific) with 5% CO2. Stromal cells were 70% confluent when lymphocytes were plated, at which point cultures were supplemented with murine IL-7 (25 ng ml21; R&D Systems) and SCF (25 ng ml21; BioLegend). For bulk cultures of PLZFhigh and CLP populations, 300–400 sorted cells were plated on OP9 stromal cells. For single-cell cultures, fetal liver cells were a4b7enriched or lineage-depleted using MACS as described previously, increasing the percentage of PLZFhigh cells from ,0.1% to up to 2% of the pre-sort population. PLZFhigh cells were then individually sorted into 96-well plates containing irradiated (1,500 rad) OP9 stromal cells. All wells were analysed after 5–6 days of culture and only wells that contained at least 8 CD451 lymphocytes were considered. To rule out the possibility of single-cell-sorting contamination by doublets, which might account for mixed ILC colonies, CD45 allele-marked fetal liver cells were mixed before sorting individual PLZFhigh cells and the monoclonal nature of CD45 allele marking was systematically verified during FACS analysis of ILC colonies. In addition, post-sorting microscopic assessment confirmed that each well of the microplate was seeded with no more than one cell. To examine the induction of GFP, 103–2.5 3 103 Lin2IL-7Ra1a4b71GFP2CXCR62 cells were sorted from adult bone marrow and plated on irradiated (1,500 rad) OP9 or OP9DL1 stromal cells. Fetal thymic organ culture (FTOC). Individual irradiated (3,000 rad) CD45.1/2 E14 fetal thymic lobes were added to wells of a Terasaki plate and suspended in a hanging drop of culture medium containing sorted BM PLZFhigh and CD45.1 CLPs (100 of each). Following a 12-h overnight culture, seeded thymic lobes were transferred to trans-well plates and cultured for 15 days before analysis. Cultures were maintained in a 37 uC incubator with 5% CO2 in RPMI 1640 containing 10% FCS (Biowest), 13 penicillin/streptomycin, and 2 mM L-glutamine (Gibco). In vivo transfers. 800–1,200 tdTomato1 PLZFhigh cells and CD45.1 CLPs were sorted and injected retro-orbitally into 6–10 week-old sub-lethally irradiated (400 rad) CD45.2 Rag22/2IL2rg2/2 mice. Recipient mice were analysed 5–7 weeks post-transfer. Bone marrow chimaeras. To remove the ‘background’ YFP staining (that is, the YFP staining occurring before the HSC stage) of bone marrow cells from PLZFGFPcre1/2 ROSA26-YFP, 5 3 103–10 3 103 YFP2Lin2Sca-11cKit1 (LSK) cells were sorted from the bone marrow of these mice and injected retro-orbitally into lethally irradiated (1,000 rad) CD45.1 recipients. The resulting chimaeras were analysed 5–7 weeks post-reconstitution by gating on CD45.21 cells to exclude residual host cells. To generate PLZF2/2/WT mixed chimaeras, 1–2 million bone marrow lymphocytes from CD45.2 PLZF2/2 and CD45.1 WT mice were injected retro-orbitally into lethally irradiated (1,000 rad) CD45.1/2 recipients. Mixed chimaeras were
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LETTER RESEARCH analysed 5–7 weeks post-reconstitution. Populations of interest were normalized to the reconstitution ratio of each chimaera, as determined by the CD45.21/ CD45.11 ratio of BM lymphocytes or splenic B cells. Quantitative PCR with reverse transcription. RNA was isolated from 5 3 103– 10 3 103 PLZFhigh, CLP, iILC2, NK and ILC3-enriched LPL using the Arcturus PicoPure RNA Isolation kit (Applied Biosystems) and reverse transcribed using the USB First-Strand cDNA Synthesis kit (Affymetrix). The following TaqMan primers (Applied Biosystems) were used (Probe ID in parenthesis): Gata3 (Mm00484683_ m1), Hprt1 (Mm00446968_m1), Id2 (Mm00711781_m1), Rora (Mm00443103_m1),
Rorc (Mm01261022_m1), Tbx21 (Mm00450960_m1), Tcf7 (Mm00493445_m1), Tox (Mm00455231_m1) and Zbtb16 (Mm01176868_m1). Quantitative PCR with reverse transcription was performed on an Mx3005p machine (Stratagene). Statistical analysis. Two-tailed Student’s t-test was performed using Prism (GraphPad Software) to determine whether data differed from the expected value. *P , 0.05; **P , 0.001; ***P , 0.0001. 29.
Lee, E. C. et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73, 56–65 (2001).
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RESEARCH LETTER
Extended Data Figure 1 | PLZF expression and lineage tracing in PLZFGFPcre mice. a, A sequence encoding an IRES and a GFP-cre fusion protein was inserted immediately after the Zbtb16 stop codon in C57BL/6J ES cells and knock-in mice were bred to ACTB-FLPe mice to excise the neomycin resistance cassette and generate the PLZFGFPcre allele. b, FACS analysis of the indicated populations from PLZFGFPcre1/2 ROSA26-YFP mice. c, Summary of data (mean 6 s.e.m.) from 2–5 mice analysed in 2 or more independent experiments.
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Extended Data Figure 2 | Gating strategy for analysis of ILC and LTi among LPL. ILC2 cells were identified as IL-7Ra1KLRG11 among CD3e2CD192 LPL (top left), and then gated Thy1.21 (not shown). CD3e2CD192 LPL were gated as IL-7Ra1KLRG12 (top left) and then subsetted into CCR61CD41 (CD41 LTi cells) and CCR61CD42 (CD42 LTi cells) (bottom left). NCR1 ILC3 were identified as CD3e2CD192 LPL that expressed NKp46 but not NK1.1 (top right).
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RESEARCH LETTER
Extended Data Figure 3 | Transcription factor expression by PLZFhigh bone marrow precursors. Quantitative PCR with reverse transcription analysis for Tbx21 and Rora as indicated. NKP are Lin2CD271IL-7Ra2Flt32CD1221 BM cells. Mean 6 s.e.m. of data from 2–3 independent experiments.
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Extended Data Figure 4 | PLZFhigh-derived NK1.11 cells are distinct from CLP-derived NK1.11 cells. CD45.2 Rag22/2Il2rg2/2 mice were injected with equivalent numbers of CD45.2 PLZFhigh cells and CD45.1 CLP (800 of each) and the resulting NK1.11CD3e2TCRb2 cells present in the spleen were analysed 5–7 weeks later by FACS, as indicated. Note that PLZFhigh-derived cells expressed higher amount of surface NKp46, whether they were identified as CD45.21 or as CD45.12 in reciprocal staining experiments. Similar results were obtained for lung NK1.11 cells. Data representative of 5 chimaeras from 2 independent experiments.
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Extended Data Figure 5 | FTOC of PLZFhigh cells. FACS analysis of PLZFhigh and CLP cells (100 of each) co-cultured for 15 days in FTOC (a). The percentages of PLZFhigh- or CLP-derived cells that are CD3e1 are summarized in the bar graph (b). Data representative of 7 independent cultures.
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Extended Data Figure 6 | Additional characterization of PLZFhigh cells after culture on OP9 cells. a, FACS analysis of PLZFhigh or CLP cells from adult BM cultured on OP9 for 4 days showing expression of T1/ST2 on ICOShigh cells. Data representative of 4 replicate cultures from 2 independent experiments. b, FACS analysis of fetal liver PLZFhigh cells after culture on OP9 for 7 days, showing expression of GATA3 by ICOShigh cells and RORct by ICOSint cells. Data representative of 2 independent experiments.
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RESEARCH LETTER
Extended Data Figure 7 | Proposed model of ILC development. A CLP-derived IL-7Ra1a4b71 population bifurcates into RORcthigh LTi precursors (LTiP) and PLZFhigh ILCP, the latter of which gives rise to all ILC lineages. Whether NKP cells develop directly from CLPs or progress through an IL-7Ra1a4b71 stage has yet to be determined.
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doi:10.1038/nature13047
A committed precursor to innate lymphoid cells Michael G. Constantinides1, Benjamin D. McDonald1, Philip A. Verhoef1 & Albert Bendelac1
1
a
CLP
B and T cells
NK T cells 3 1 and
iILC2
ILC2
2
GFP 0
0
94
69
80
YFP
b
NK cells
DX5+ cells
DX5– cells
ILC1
GFP 22
17
74
CD4+ LTi
CD4– LTi
NCR+ ILC3
13
60
68
YFP
c
GFP 4
YFP
d Per cent YFP+
100 75 50 25 0
BM Lu iIL ng C2 LP L ILC L PL 2 N IL C LP R + C2 L IL LP CD C3 L 4– C LT D4 i + IE LT L Li IL i ve C 1 Li r DX ve 5 – r Sp DX le 5 + en Sp N le K Sp en Sp le B le en en T N K T
Innate lymphoid cells (ILCs) specialize in the rapid secretion of polarized sets of cytokines and chemokines to combat infection and promote tissue repair at mucosal barriers1–9. Their diversity and similarities with previously characterized natural killer (NK) cells and lymphoid tissue inducers (LTi) have prompted a provisional classification of all innate lymphocytes into groups 1, 2 and 3 solely on the basis of cytokine properties10, but their developmental pathways and lineage relationships remain elusive. Here we identify and characterize a novel subset of lymphoid precursors in mouse fetal liver and adult bone marrow that transiently express high amounts of PLZF, a transcription factor previously associated with NK T cell development11,12, by using lineage tracing and transfer studies. PLZFhigh cells were committed ILC progenitors with multiple ILC1, ILC2 and ILC3 potential at the clonal level. They excluded classical LTi and NK cells, but included a peculiar subset of NK1.11DX52 ‘NK-like’ cells residing in the liver. Deletion of PLZF markedly altered the development of several ILC subsets, but not LTi or NK cells. PLZFhigh precursors also expressed high amounts of ID2 and GATA3, as well as TOX, a known regulator of PLZF-independent NK and LTi lineages13. These findings establish novel lineage relationships between ILC, NK and LTi cells, and identify the common precursor to ILCs, termed ILCP. They also reveal the broad, defining role of PLZF in the differentiation of innate lymphocytes. To study the expression pattern of Zbtb16 encoding the transcription factor PLZF, which directs the developmental acquisition of the innate effector program of NK T cells11,12,14,15, we inserted a sequence coding for a fusion of enhanced green fluorescent protein (GFP) and Cre downstream of an IRES after the last exon of Zbtb16 (Extended Data Fig. 1a). As expected, GFP was selectively expressed in the NK T lineage, with early developmental stages 1 and 2 showing higher levels than mature stage 3 cells, but was not found in the bone marrow common lymphoid precursor (CLP), T cells or B cells of PLZFGFPcre1/2 mice (Fig. 1a). In PLZFGFPcre1/2 mice carrying the ROSA26-floxstopyellow fluorescent protein (YFP) fate-mapping allele, nearly all NK T cells expressed YFP, as expected, although approximately 35% of cells in all lymphoid and myeloid lineages were also labelled (Extended Data Fig. 1b, c and data not shown). Because haematopoietic stem cells (HSC) did not express GFP but were already labelled by YFP, this ‘background’ reflected some expression of PLZF before the HSC stage, probably in multipotent embryonic cells. Indeed, after transfer of fluorescenceactivated cell sorting (FACS)-sorted YFP-negative bone marrow cells into lethally irradiated recipients, 94% of NK T cells still expressed YFP, whereas donor-derived CLPs, B cells and T cells were unlabelled (Fig. 1a). Thus, the experiments shown in Fig. 1 were conducted with such bone marrow chimaeras, although all results were confirmed in non-chimaeric reporter mice. Intriguingly, several innate lymphoid lineages, which arise from CLP, were also specifically labelled by YFP despite absence of GFP expression. Thus, ILC2 in the lungs, intestinal lamina propria (LP), peritoneal cavity and mesenteric lymph nodes were all fate-mapped (Fig. 1a, d, Extended Data Fig. 1b and data not shown). Immature ILC2 in the bone marrow (iILC2) expressed very low amounts of GFP, but were already maximally labelled by YFP, suggesting expression of a higher level of PLZF at an earlier stage of their development (Fig. 1a, d). Group 1 innate lymphocyte subsets showed heterogeneous PLZF tracing:
Figure 1 | ILC lineage tracing in PLZFGFPcre reporter mice. a–c, Top rows, expression of GFP by indicated cell-types of PLZFGFPcre1/1 mice (filled grey) and wild type (WT; open). Bottom rows, YFP expression in radiation chimaeras reconstituted with YFP2Lin2Sca-11cKit1 (LSK) bone marrow cells from PLZFGFPcre1/2 ROSA26-YFP mice. CLP and iILC2 from bone marrow (BM); B and T cells and NK cells from spleen; NK T cells from thymus (GFP), divided into early stages 1, 2 and late stage 3, and from spleen (YFP); ILC2 from lung; DX51 and DX52 NK cells from liver; ILC1 from intestinal intraepithelial lymphocytes (IEL); LTi and ILC3 from LPL. BM iILC2 and lung ILC2 were identified as Lin2CD251IL-7Ra1Thy1.21; LPL ILC2 as CD3e2CD192 CD251KLRG11; LPL NCR1 ILC3 as CD3e2CD192NKp461NK1.12; LPL CD42 LTi cells as CD3e2CD192IL-7Ra1KLRG12CCR61CD42; LPL CD41 LTi cells as CD3e2CD192CCR61CD41; and IEL ILC1 as CD3e2CD192 NKp461NK1.11CD1601. FACS identification of the remaining subsets is defined in the methods section. d, Summary of results (mean 6 s.e.m.). Data representative of 3–9 individual mice analysed in at least 2 independent experiments.
Committee on Immunology, Department of Pathology, The Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, USA. 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 1
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RESEARCH LETTER whereas few splenic NK cells expressed YFP, intestinal intraepithelial NK-like cells (termed ILC11) were prominently labelled (Fig. 1b). In the liver the recently described non-recirculating DX52CD49a1 subset of CD3e2NK1.11 cells16 was heavily labelled, whereas classical recirculating DX51CD49a2 NK cells were mostly negative. Different subsets of group 3 innate lymphocytes in the LP also showed markedly different patterns of tracing (Fig. 1c and Extended Data Fig. 2). CD41 and CD42 LTi were not labelled, whereas NCR1 ILC3 prominently expressed YFP. In summary, PLZF lineage-tracing labelled not only ILC2 but also the subsets of group 1 and group 3 cells that are most clearly distinguishable from classical NK and LTi cells, respectively, and will hereafter be termed ILC1 and ILC3. Searching for the PLZF-expressing precursor of ILCs, we identified a rare subset of PLZFhigh cells in fetal liver and adult bone marrow. They showed a homogeneous lineage2(Lin2)IL-7Ra1cKit1a4b7high phenotype (Fig. 2a, b), similar to the CLP-derived subset previously suggested to contain precursors for LTi17–20. The PLZFhigh population represented approximately 5% of Lin2IL-7Ra1cKit1 cells and approximately 30% of the a4b7high fraction (Fig. 2b, c). It expressed Thy1 but lacked expression of markers associated with mature ILCs, NK or LTis such as CD4, CXCR6, CD25, IL-17RB, T1/ST2, Sca-1, CD122, NK1.1, CCR6 and NKp46 (Fig. 2c, d and data not shown). Interestingly, the PLZFhigh cells included a fraction of CD62LhighICOSlow cells, probably representing the earliest developmental stage after PLZF expression, because PLZF characteristically induces the downregulation of CD62L and upregulation of ICOS15,21. Transcriptional analysis of purified a
b Lin– IL-7Rα+ cKit+ E14 fetal liver d
Adult bone marrow PLZFGFPcre+/+
WT
PLZFGFPcre+/–
WT 12
8
0
CD25
CD44
CD62L
ICOS
IL-17RB
Sca-1
T1/ST2
Thy1.2
4
0.01
α4β7
Lineage
0
PLZFhigh cells compared with bone marrow CLP, iILC2 and NK progenitors (NKP), and with ILC3-enriched lamina propria lymphocytes (LPL), confirmed the very high amounts of Zbtb16 mRNA encoding PLZF, as well as of other key transcription factors such as Id2, Gata3 and Rora, which are required for ILC2 development3,22–25 (Fig. 2e and Extended Data Fig. 3) and Tcf7, a target of Notch required for ILC2 and ILC326,27. Notably, they expressed very high amounts of Tox, which is required for the development of both NK and LTi cells13, and low but detectable amounts of Tbx21 and Rorc, the transcription factors associated with ILC1 and ILC3, respectively1,6. Single-cell analysis by intracellular flow cytometry was performed after MACS-depletion of Lin1 cells in fetal liver and adult bone marrow, gating on Lin2IL-7Ra1 a4b7high cells (Fig. 2f). PLZFhigh cells, which represented up to 40% of this population, characteristically co-expressed high amounts of both GATA3 and TOX. Interestingly, a small fraction of these PLZFhigh cells also expressed RORct, although these were mostly found in the fetal liver and were rare in adult bone marrow. A distinct population consisting of RORcthigh PLZF2 cells, which was more abundant in the fetal liver, co-expressed high levels of TOX but not GATA3, probably representing LTi precursors. Finally, a fraction of Lin2IL-7Ra1a4b7high cells lacked both PLZF and RORct, but expressed TOX. These may be earlier undifferentiated precursors to the ILC and LTi lineages, and may also include precursors to classical NK cells. To determine precursor-product relationships, we injected a 1:1 mixture of PLZFhigh cells and CLP cells into Rag22/2Il2rg2/2 mice. The PLZFhigh cells, which were purified from a ROSA-floxstop-tdTomato 3
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Figure 2 | PLZFhigh cells in fetal liver and bone marrow. a, FACS analysis of total BM cells from adult WT littermate and PLZFGFPcre1/1 mice. Data representative of at least 3 independent experiments. b, c, GFP expression is confined to the a4b7high population of indicated fractions in fetus and adult. Representative of at least 3 independent experiments. d, FACS analysis of PLZFhigh BM cells (solid line) and BM iILC2 (dashed line) compared with isotype controls (filled). iILC2 were gated as Lin2ICOS1T1/ST21Sca-11Thy1.21 for CD25 profile, as CD251CD62L2Sca-11Thy1.21 for CD44 profile, as CD251CD441Sca-11Thy1.21 for CD62L profile, as CD251T1/ST21Sca11Thy1.21 for ICOS profile, as CD251ICOS1Sca11Thy1.21 for T1/ST2 profile, as CD251Thy1.21 for Sca-1 profile or as CD251ICOS1Sca-11 for Thy1.2 profile. Representative of at least 3 independent experiments. e, Quantitative PCR with reverse transcription analysis in bone marrow subsets as indicated. NKP are Lin2CD271IL7Ra2Flt32CD1221 BM cells and ILC3-enriched LPL are CD3e2CD192IL7Ra1Thy1.21KLRG12CD42 cells. Mean 6 s.e.m. of data from 2–3 independent experiments. f, Intracellular FACS analysis for transcription factors in cells as indicated. Representative of at least 3 independent experiments.
LETTER RESEARCH PLZFGFPcre1/2 background to further ascertain their history of PLZF expression based on tdTomato expression, gave rise to a diversity of innate lineages, with the notable exception of CD41 LTi (Fig. 3a). Significantly more peripheral ILC2 were derived from the PLZFhigh b
CD45.1
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BM Lu iILC ng 2 RO IL Rγ L t + PL C2 LP NC IL LP L R + C2 R L RO OR (ILC Rγ γt + 3) t+ N C CR D4 – + ( IE LTi) L Li ILC ve Lu r 1 L DX n Sp g N ive 5 – le K1 r D en .1 + X5 + N K1 CD .1 + 3ε – C D Sp 3ε – le e Sp n B le en T
6.8
Per cent PLZFhigh-derived
a
fraction at 5–7 weeks post-injection (Fig. 3a, c). Both progenitors gave rise to similar amounts of CD3e2NK1.11 group 1 cells, but CLP mostly generated the classical DX51CD49a2NK1.11 NK cells, whereas the PLZFhigh cells primarily generated a distinct DX5–CD49a1NK1.11 ILC1
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Figure 3 | PLZF cells are ILC progenitors. a–c, CD45.2 Rag2 Il2rg mice were injected with equivalent numbers of tdTomato1 PLZFhigh cells and CD45.1 CLP (800–1,200 of each) and the progeny of these populations analysed 5–7 weeks later by FACS, as indicated. Summary bar graph of mean percentages 6 s.e.m. in c, with significant differences between PLZFhigh- (black bar) and CLP-derived (white bar) shown by *. LPL RORct1 NCR1 cells were identified as CD3e2CD192RORct1NKp461CD42; LPL RORct1NCR2 cells as CD3e2CD192RORct1NKp462CD42; and LPL RORct1CD41 cells as CD3e2CD192RORct1CD41. Remaining populations were gated as indicated in Fig. 1. Data representative of 6–9 chimaeras analysed in at least 2 independent experiments. d, FACS analysis of Lin2IL7Ra1a4b7highGFP2CXCR62 cells sorted from PLZFGFPcre1/2 BM and cultured on OP9 or OP9-DL1 for 48 h. Data representative of 3 independent experiments. e, FACS analysis of sorted PLZFhigh BM cells cultured on OP9 for indicated periods. Data representative of at least 4 replicate cultures for each time point from 2 or more independent experiments. f, FACS analysis of
ILC Number of colonies type Exp 1 Exp 2 Exp 3 Exp 4 1 1,2,3 2 0 4 0 1,2 3 2 4 2 1,3 6 3 10 10 2,3 10 0 1 45 ILC1 50 35 100 42 ILC2 30 16 53 ILC3 3 11 35 22
Total 7 9 21 21 230 141 71
Colony size 200 ± 32 156 ± 32 134 ± 23 96 ± 15 72 ± 8 33 ± 2 49 ± 7
PLZFhigh or CLP cells from adult BM or fetal liver cultured on OP9 for 4 days. Data representative of at least 4 replicate cultures from 2 or more independent experiments. g, FACS analysis of representative colonies originating from single fetal liver PLZFhigh cells that were sorted and cultured into 96-well plates containing irradiated OP9 stromal cells for 5–6 days. ILC1 were characterized as ICOSlowa4b72 NK1.11 populations, ILC2 as ICOShigha4b72NK1.12, and ILC3 as ICOSint/higha4b71NK1.12. The table summarizes the analysis of thirteen 96-well plates analysed in four independent experiments (three plates in experiments 1, 2 and 4; four plates in experiment 3), with the average colony size 6 s.e.m. as indicated. In experiment 4, we mixed a 1:1 ratio of CD45.1/5.2 and CD45.2 PLZFGFPcre1/2 fetal liver cells before single-cell sorting. All 122 colonies were either CD45.1/5.2 (n 5 59) or CD45.2 (n 5 63), ruling out doublet contamination as an explanation for the presence of mixed ILC colonies (P , 0.01 Chi-square). The cloning efficiency was 40% on average, with all but 17 colonies (not shown in the table) unambiguously assigned to defined ILC lineages. 0 0 M O N T H 2 0 1 4 | VO L 0 0 0 | N AT U R E | 3
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0.0 BM i LP Lu ILC L ng 2 RO IL Rγ C L t + PL 2 N LP C ILC R+ 2 LP L R ( L RO OR ILC Rγ γt + 3) t + NC C D4 R – + (L IE Ti) L I L Li ve C1 r Li DX5 ve – rD X5 +
subset in the liver (Fig. 3b, c). PLZFhigh-derived NK1.11CD3e–TCRb– cells were observed in the spleen and lungs, and expressed significantly more NKp46 than CLP-derived NK cells (Extended Data Fig. 4). Unlike CLP, PLZFhigh cells did not generate B cells, consistent with their expression of Tcf7, suggesting that they had already received a Notch signal in vivo. They also gave rise to far fewer T cells than CLP after transfer into Rag22/2Il2rg2/2 mice in vivo (Fig. 3a, c) or after coculture with fetal thymic stroma in vitro (Extended Data Fig. 5), indicating that PLZFhigh cells had also largely lost T cell potential. Thus, the PLZFhigh cell is an ILC precursor (ILCP) heavily committed to innate lymphoid lineages, excluding classical NK and LTi cells. When cultured on OP9 stromal cells, the GFP2 fraction of Lin2IL7Ra1cKit1a4b7high cells generated a small fraction of GFP1 cells within 2 days of culture, which was increased upon culture on OP9DL1 cells (Fig. 3d), confirming the precursor–product relationship and its promotion by Notch signals. To study the short-term fate of ILCP in vitro, we sorted Lin2IL-7Ra1cKit1a4b7high PLZFhigh cells from either adult bone marrow or fetal liver and cultured them on OP9 stromal cells, without Notch ligands, in the presence of non-polarizing IL-7 and stem-cell factor (SCF). By 24 h, a substantial fraction of the PLZFhigh population had already begun to downregulate GFP and to upregulate either ICOS or CD122 (Fig. 3e), and by 4 days, the cells had resolved into three separate populations of ILC1 (ICOS2IL-17RB2T1/ ST22CD252RORct2GATA32a4b72CD1221NK1.11NKp461), ILC2 (ICOShighIL-17RB1T1/ST21CD251RORct2GATA31a4b72CD1222 NK1.12NKp462), and ILC3 (ICOSintIL17RB2T1/ST22CD252RORct1 GATA32a4b71CD1222NK1.12NKp462/low) (Fig. 3f and Extended Data Fig. 6). This transient expression of PLZF before ILC differentiation may explain why fate-mapping by the ROSA-YFP allele in Fig. 1 did not quite reach 100% for these populations as in NK T cells where PLZF expression is permanent. Notably, ILC3 cells consistently arose with higher frequency from fetal liver than from adult bone marrow cells (Fig. 3f). In single-cell cultures with OP9 stromal cells, fetal PLZFhigh cells generated mixtures of ILC lineages, including triple and double ILC1, ILC2 and ILC3, in 58 out of 500 wells (12%) as well as single ILC1, ILC2 or ILC3 populations in the remaining wells (Fig. 3g). Mixing CD45 allele-marked fetal liver cells before single-cell sorting confirmed that the mixed ILC colonies did not result from cell aggregates escaping exclusion during cell-sorting (Fig. 3g, experiment 4) and post-sorting microscopic assessment confirmed single-cell seeding in each of 96 wells. These results indicated that the PLZFhigh cells contained a common ILC progenitor characterized by multiple ILC lineage potential at the clonal level, and that subsequent commitment to individual ILC1, ILC2 or ILC3 lineages occurred rapidly during the PLZFhigh stage of ILC development. Wells containing mixtures of ILC lineages had more growth than those with single lineages, indicating that multipotent ILC progenitors retained greater proliferative capacity. We then assessed the contribution of PLZF to ILC development in lethally irradiated recipients of a mixture of bone marrow from congenically labelled Zbtb162/2 and Zbtb161/1 littermate donors (Fig. 4a). The production of ILC2 was markedly decreased in the PLZF-deficient compartment of all tissues, demonstrating a cell-intrinsic requirement of PLZF. Furthermore, residual immature ILC2 in the bone marrow expressed significantly less ICOS and more CD62L in the absence of PLZF (Fig. 4b, c), a phenotype entirely consistent with that of PLZFdeficient NK T cells11,12,15, which accounts for altered recirculating properties and reduced frequencies in peripheral tissues. LTi cells were unaffected by the genetic deletion of PLZF, as expected (Fig. 4a). Other ILC3 cells, including NCR1 ILC3, were unaffected, despite their expression of PLZF during development. However, liver ILC1 (DX52CD49a1) were profoundly affected, similar to ILC2, whereas classical (DX51CD49a2) NK cells were not (Fig. 4a). Our studies have identified the elusive CLP-derived common progenitor to the ILC1, ILC2, and ILC3 lineages and demonstrated close but distinct lineage relationships with classical NK and LTi cells. We have shown that a committed PLZFhigh ILCP emerges from within the
Figure 4 | PLZF is required for ILC development. Lethally irradiated CD45.1/2 mice were reconstituted with a mixture of CD45.2 Zbtb162/2 and CD45.1 WT BM and analysed as indicated 5–7 weeks later. a, Representative FACS analysis of LPL gated as indicated, and summary bar graph of mean percentages 6 s.e.m. for indicated populations, with significant variation from 1.00 indicated by *. Populations were gated as indicated in Fig. 3. Data representative of 6–10 chimaeras analysed in at least 2 independent experiments. b, c, FACS analysis of iILC2 from Zbtb162/2 mice and Zbtb161/1 littermates, representative of 5 (b) and 8 (c) mice of each genotype analysed in 3 or more independent experiments.
same IL-7Ra1a4b7high population that includes precursors to LTi and possibly also to NK cells (Extended Data Fig. 7). It is uniquely distinguished and affected by the rapid and transient upregulation of PLZF, whereas TOX is more broadly induced and reciprocally controls classical NK and LTi cells. The concomitant induction of GATA3, RORa, Tbet and RORct, which govern the polarization of ILCs, parallels the sequence during PLZF-dependent NK T cell differentiation28, indicating a broad and defining role of PLZF in the differentiation of innate lymphocyte lineages.
METHODS SUMMARY PLZFGFPcre mice were generated by inserting a sequence encoding an IRES and EGFP–Cre fusion protein immediately after the Zbtb16 stop codon through homologous recombination in C57BL/6J embryonic stem cells. For fate-mapping, they were crossed to mice carrying a ROSA26-fl-STOP-fl-YFP or a ROSA26-fl-STOPfl-tdTomato allele. Bone marrow chimaeras were generated by reconstituting lethally irradiated recipients with purified Lin2 Sca-11 cKit1 (LSK) bone marrow cells. PLZFhigh ILC precursors were purified from bone marrow or fetal liver and cultured in bulk or as single cells deposited by flow cytometry in 96-well plates with irradiated OP9 or OP9-DL1 stromal cells and murine IL-7 and SCF. For in vivo transfers, PLZFhigh ILC precursors and CLP were purified from bone marrow and injected into Rag22/2IL2rg2/2 mice. Quantitative PCR with reverse transcription for transcription factors was performed using TaqMan primers. Online Content Any additional Methods, Extended Data display items and Source Data are available in the online version of the paper; references unique to these sections appear only in the online paper. Received 27 November 2013; accepted 21 January 2014. Published online 9 February 2014. 1. 2. 3.
Fuchs, A. et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL12- and IL-15-responsive IFN-c-producing cells. Immunity 38, 769–781 (2013). Neill, D. R. et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464, 1367–1370 (2010). Moro, K. et al. Innate production of TH2 cytokines by adipose tissue-associated c-Kit1Sca-11 lymphoid cells. Nature 463, 540–544 (2010).
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LETTER RESEARCH 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
18.
Price, A. E. et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proc. Natl Acad. Sci. USA 107, 11489–11494 (2010). Monticelli, L. A. et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nature Immunol. 12, 1045–1054 (2011). Satoh-Takayama, N. et al. Microbial flora drives interleukin 22 production in intestinal NKp461 cells that provide innate mucosal immune defense. Immunity 29, 958–970 (2008). Sanos, S. L. et al. RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp461 cells. Nature Immunol. 10, 83–91 (2009). Luci, C. et al. Influence of the transcription factor RORct on the development of NKp461 cell populations in gut and skin. Nature Immunol. 10, 75–82 (2009). Cella, M. et al. A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature 457, 722–725 (2009). Spits, H. et al. Innate lymphoid cells–a proposal for uniform nomenclature. Nature Rev. Immunol. 13, 145–149 (2013). Savage, A. K. et al. The transcription factor PLZF directs the effector program of the NKT cell lineage. Immunity 29, 391–403 (2008). Kovalovsky, D. et al. The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions. Nature Immunol. 9, 1055–1064 (2008). Aliahmad, P., de la Torre, B. & Kaye, J. Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages. Nature Immunol. 11, 945–952 (2010). Savage, A. K., Constantinides, M. G. & Bendelac, A. Promyelocytic leukemia zinc finger turns on the effector T cell program without requirement for agonist TCR signaling. J. Immunol. 186, 5801–5806 (2011). Constantinides, M. G., Picard, D., Savage, A. K. & Bendelac, A. A naive-like population of human CD1d-restricted T cells expressing intermediate levels of promyelocytic leukemia zinc finger. J. Immunol. 187, 309–315 (2011). Peng, H. et al. Liver-resident NK cells confer adaptive immunity in skin-contact inflammation. J. Clin. Invest. 123, 1444–1456 (2013). Yoshida, H. et al. Expression of a4b7 integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells. J. Immunol. 167, 2511–2521 (2001). Sawa, S. et al. Lineage relationship analysis of RORct1 innate lymphoid cells. Science 330, 665–669 (2010).
19. Possot, C. et al. Notch signaling is necessary for adult, but not fetal, development of RORct1 innate lymphoid cells. Nature Immunol. 12, 949–958 (2011). 20. Cherrier, M., Sawa, S. & Eberl, G. Notch, Id2, and RORct sequentially orchestrate the fetal development of lymphoid tissue inducer cells. J. Exp. Med. 209, 729–740 (2012). 21. Gleimer, M., von Boehmer, H. & Kreslavsky, T. PLZF controls the expression of a limited number of genes essential for NKT cell function. Front. Immunol. 3, 374 (2012). 22. Hoyler, T. et al. The transcription factor GATA-3 controls cell fate and maintenance of type 2 innate lymphoid cells. Immunity 37, 634–648 (2012). 23. Mjo¨sberg, J. et al. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. Immunity 37, 649–659 (2012). 24. Wong, S. H. et al. Transcription factor RORa is critical for nuocyte development. Nature Immunol. 13, 229–236 (2012). 25. Halim, T. Y. et al. Retinoic-acid-receptor-related orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation. Immunity 37, 463–474 (2012). 26. Yang, Q. et al. T cell factor 1 is required for group 2 innate lymphoid cell generation. Immunity 38, 694–704 (2013). 27. Mielke, L. A. et al. TCF-1 controls ILC2 and NKp461RORct1 innate lymphocyte differentiation and protection in intestinal inflammation. J. Immunol. 191, 4383–4391 (2013). 28. Constantinides, M. G. & Bendelac, A. Transcriptional regulation of the NKT cell lineage. Curr. Opin. Immunol. 25, 161–167 (2013). Acknowledgements We thank W. Yokoyama for discussion; F. Gounari and R. de Pooter for advice on OP9 cultures; H. Gudjonson for statistical advice; D. Leclerc, J. Cao, M. Olsen and R. Duggan for help with cell sorting; V. Bindokas and R. Mathew for help with fluorescence microscopy. This work was supported by NIH grants R01HL118092, R01AI038339 and P30DK42086 and by The Howard Hughes Medical Institute (A.B.). Author Contributions M.G.C., B.D.M. and P.A.V. designed research, performed experiments and analysed data. M.G.C. and A.B. wrote the paper. A.B. supervised the research. Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to A.B. ([email protected]).
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RESEARCH LETTER METHODS a
b
Mice. C57BL/6J (stock no. 000664), B6.SJL-Ptprc Pepc /BoyJ (CD45.1; stock no. 002014), B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (stock no. 006148), B6.CgGt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (stock no. 007914), and B6.Cg-Tg(ACTFLPe) 9205Dym/J (stock no. 005703) mice were obtained from The Jackson Laboratory, whereas B6.Rag2tm1Fwa II2rgtm1Wjl (stock no. 4111) mice were obtained from Taconic. Plzf2/2 mice were a gift from P. P. Pandolfi and were backcrossed to C57BL/6J for at least nine generations. Animals were 4–10 weeks of age when analysed and were compared to littermate controls. For fetal experiments, the morning a vaginal plug was observed was considered embryonic day 0 (E0). Mice were housed in a specific-pathogen-free environment at the University of Chicago and experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. Generation of the PLZFGFPcre reporter strain. The PLZFGFPcre strain was generated as follows. A sequence encoding an IRES and GFP–Cre fusion protein was cloned from the pIGCN21 plasmid29 and inserted immediately following the Zbtb16 stop codon. The linearized construct was transfected into C57BL/6J embryonic stem cells and neomycin-resistant clones were screened by PCR. Clones that had undergone homologous recombination were injected into albino C57BL/6J blastocysts and the resulting chimaeric mice were crossed with ACTB-FLPe mice to excise the neomycin resistance cassette. The following PCR primers were used to identify WT (296 base pairs) and knock-in (419 base pairs) alleles: 59-GTGTACC ATCTGCACGGAAT-39, 59-GGCCAAGTAACTATCAGGCT-39, and 59-TACC GGAGATCATGCAAGCT-39. Preparation of cell suspensions. Spleen, liver and lymph nodes were mechanically dissociated through 70-mm filters and bone marrow was isolated by gently crushing femurs and tibias before filtration. Following dissociation, each liver was centrifuged at 400g for 5 min, resuspended in 5 ml of 40% Percoll (Sigma-Aldrich), and then centrifuged at 800g for 10 min. The supernatant was aspirated and the cell pellet was resuspended in HBSS (Gibco) containing 0.25% BSA (Sigma-Aldrich) and 0.65 mg l21 sodium azide (Sigma-Aldrich). For the isolation of lung lymphocytes, mice were anaesthetized with ketamine/ xylazine and approximately 1 ml of PBS (Sigma-Aldrich) was injected into the right ventricle to perfuse the lung tissue. Pairs of lungs were diced and incubated in 5 ml of pre-warmed RPMI 1640 (Cellgro) containing 0.01% DNase I (Roche), and 650 units per ml collagenase I (Worthington) in a 37 uC shaking incubator for 30 min. The digested tissue was passed through a 70-mm filter and centrifuged at 400g for 5 min. The cells were resuspended in 5 ml of 40% Percoll, underlaid with 5 ml of 60% Percoll, and centrifuged at 800g for 20 min with no brake. Lymphocytes were isolated from the interface and resuspended in HBSS containing 0.25% BSA and 0.65 mg l21 sodium azide. To isolate IEL and LPL, the small intestine was emptied of its contents, opened by making a lengthwise incision, and cut into 1-cm pieces which were then shaken at 37 uC for 30 min in RPMI 1640 containing 1% FCS (Biowest), 1 mM EDTA (Fluka), and 1 mM DTT (Sigma-Aldrich). Following the incubation, the supernatant was centrifuged at 400g for 5 min, resuspended in 5 ml of 40% Percoll, and then centrifuged at 2,000g for 10 min, yielding the IEL fraction. The pieces of tissue were subjected to two 30-min digestions in RPMI 1640 containing 20% FCS, 0.2 mg ml21 DNase I, and 100 units ml21 collagenase VIII (Sigma-Aldrich). The digested tissue was passed through a 70-mm filter, centrifuged at 400g for 5 min, resuspended in 5 ml of 40% Percoll, and then centrifuged at 2,000g for 10 min to isolate the LPL fraction. Flow cytometry. Cell suspensions were incubated with purified anti-CD16/32 (clone 93) for 10 min on ice to block Fc receptors. Fluorochrome- or biotin-labelled monoclonal antibodies (clones denoted in parenthesis) against 2B4 (2B4), a4b7 (DATK32), B220 (RA3-6B2), CCR6 (29-2L17), CD3 (17A2), CD3e (145-2C11), CD4 (RM4-5 or GK1.5), CD8a (53-6.7), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD25 (PC61), CD27 (LG.7F9), CD44 (IM7), CD45.1 (A20), CD45.2 (104), CD49a (HMa1), CD62L (MEL-14), CD122 (5H4 or TM-b1), CD160 (7H1), cKit (2B8), CXCR6 (221002), DX5 (DX5), Flt3 (A2F10), Gr-1 (RB6-8C5), I-A/I-E (M5/ 114.15.2), ICOS (C398.4A), IL-17RB (752101), IL-7Ra (A7R34), KLRG1 (2F1), NK1.1 (PK136), NKp46 (29A1.4), Sca-1 (D7), T1/ST2 (D1H9), TCRb (H57-597), Ter-119 (TER-119), and Thy1.2 (53-2.1) were purchased from BD Biosciences, BioLegend, eBioscience or R&D Systems. CD1d-PBS57 tetramer was from the NIH tetramer facility. For pre-enrichment of fetal liver and BM PLZFhigh cells, samples were stained with allophycocyanin (APC)-conjugated anti-a4b7 antibody, bound to anti-APC microbeads (Miltenyi Biotec), and subjected to doublecolumn enrichment on an autoMACS (Miltenyi Biotec). For the isolation of HSC, CLP, BM iILC2 and lung ILC2, lineage1 cells were depleted by staining with biotin-conjugated antibodies against B220, CD3e, CD4, CD8a, CD11b, CD11c, CD19, Gr-1, NK1.1, TCRb and Ter119, followed by an incubation with SAv microbeads (Miltenyi Biotec). Fetal liver progenitors were similarly enriched, except the biotin-conjugated lineage antibodies used were against B220, CD3e, CD11c, CD19,
NK1.1, NKp46 and Ter119. To exclude dead cells, 49,6-diamidino-2-phenylindole (DAPI; Molecular Probes) was added to all live samples. Cells were run on an LSRII (BD Biosciences) or sorted using a FACS Aria II (BD Biosciences) and analysed using FlowJo software (Tree Star). Collected events were gated on DAPI2 lymphocytes and doublets were excluded. For the intracellular staining of fetal liver progenitors, sorted Ly5.2 Lin2IL7Ra1a4b71 cells were combined with 2 3 105 Ly5.1 thymocytes to increase the size of the cell pellet. Samples were stained with anti-CD45.1 and anti-CD45.2 antibodies and fixed and permeabilized using the Foxp3 staining kit (eBioscience). Samples were then stained with monoclonal antibodies against GATA3 (TWAJ), RORct (B2D), and/or Tox (TXRX10). Intracellular PLZF was detected by conjugating a purified monoclonal antibody (D9; Santa Cruz) to Alexa Fluor 647 using an antibody labelling kit (Molecular Probes). Unless otherwise indicated, PLZFhigh cells were identified and sorted as Lin2IL7Ra1a4b71EGFP1; CLP as Lin2IL-7Ra1cKitintSca-1intFlt3high; HSC as Lin2 cKit1Sca-11Flt32; BM iILC2 and lung ILC2 as Lin2CD251IL-7Ra1Thy1.21; LPL ILC2 as CD3e2CD192CD251KLRG11; LPL NCR1 ILC3 as CD3e2CD192 NKp461NK1.12; LPL RORct1 NCR1 cells as CD3e2CD192RORct1NKp461 CD42; LPL RORct1 NCR2 cells as CD3e2CD192RORct1NKp462CD42; LPL CD41 LTi cells as CD3e2CD192CCR61CD41; LPL RORct1 CD41 cells as CD3e2CD192RORct1CD41; LPL CD42 LTi cells as CD3e2CD192IL7Ra1KLRG12CCR61CD42; IEL ILC1 as CD3e2CD192NKp461NK1.11CD1601; liver DX51 cells as CD3e2TCRb2NK1.11DX51CD49a2; liver DX52 cells as CD3e2TCRb2NK1.11DX52CD49a1; spleen NK cells as CD3e2NK1.11; spleen DC as CD11c1I-A/I-E1; spleen T cells as TCRb1; spleen B cells as CD191; NK T cells as CD1d-PBS57 tetramer1 CD242 in the thymus and CD1d-PBS57 tetramer1 TCRb1 in the spleen. Lineage antibodies against the following markers were used: B220, CD3e, CD4, CD8a, CD11b, CD11c, CD19, Gr-1, NK1.1, TCRb and Ter119. OP9/OP9-DL1 cultures. Stocks of OP9 and OP9-DL1 stromal cells were a gift from J. C. Zu´n˜iga-Pflu¨cker. All experiments were performed in Opti-MEM with GlutaMAX (Gibco) containing 10% FCS (Gibco), 13 penicillin/streptomycin (Gibco), and 60 mM 2-mercaptoethanol (Sigma-Aldrich) and maintained in a 37 uC incubator (Thermo Scientific) with 5% CO2. Stromal cells were 70% confluent when lymphocytes were plated, at which point cultures were supplemented with murine IL-7 (25 ng ml21; R&D Systems) and SCF (25 ng ml21; BioLegend). For bulk cultures of PLZFhigh and CLP populations, 300–400 sorted cells were plated on OP9 stromal cells. For single-cell cultures, fetal liver cells were a4b7enriched or lineage-depleted using MACS as described previously, increasing the percentage of PLZFhigh cells from ,0.1% to up to 2% of the pre-sort population. PLZFhigh cells were then individually sorted into 96-well plates containing irradiated (1,500 rad) OP9 stromal cells. All wells were analysed after 5–6 days of culture and only wells that contained at least 8 CD451 lymphocytes were considered. To rule out the possibility of single-cell-sorting contamination by doublets, which might account for mixed ILC colonies, CD45 allele-marked fetal liver cells were mixed before sorting individual PLZFhigh cells and the monoclonal nature of CD45 allele marking was systematically verified during FACS analysis of ILC colonies. In addition, post-sorting microscopic assessment confirmed that each well of the microplate was seeded with no more than one cell. To examine the induction of GFP, 103–2.5 3 103 Lin2IL-7Ra1a4b71GFP2CXCR62 cells were sorted from adult bone marrow and plated on irradiated (1,500 rad) OP9 or OP9DL1 stromal cells. Fetal thymic organ culture (FTOC). Individual irradiated (3,000 rad) CD45.1/2 E14 fetal thymic lobes were added to wells of a Terasaki plate and suspended in a hanging drop of culture medium containing sorted BM PLZFhigh and CD45.1 CLPs (100 of each). Following a 12-h overnight culture, seeded thymic lobes were transferred to trans-well plates and cultured for 15 days before analysis. Cultures were maintained in a 37 uC incubator with 5% CO2 in RPMI 1640 containing 10% FCS (Biowest), 13 penicillin/streptomycin, and 2 mM L-glutamine (Gibco). In vivo transfers. 800–1,200 tdTomato1 PLZFhigh cells and CD45.1 CLPs were sorted and injected retro-orbitally into 6–10 week-old sub-lethally irradiated (400 rad) CD45.2 Rag22/2IL2rg2/2 mice. Recipient mice were analysed 5–7 weeks post-transfer. Bone marrow chimaeras. To remove the ‘background’ YFP staining (that is, the YFP staining occurring before the HSC stage) of bone marrow cells from PLZFGFPcre1/2 ROSA26-YFP, 5 3 103–10 3 103 YFP2Lin2Sca-11cKit1 (LSK) cells were sorted from the bone marrow of these mice and injected retro-orbitally into lethally irradiated (1,000 rad) CD45.1 recipients. The resulting chimaeras were analysed 5–7 weeks post-reconstitution by gating on CD45.21 cells to exclude residual host cells. To generate PLZF2/2/WT mixed chimaeras, 1–2 million bone marrow lymphocytes from CD45.2 PLZF2/2 and CD45.1 WT mice were injected retro-orbitally into lethally irradiated (1,000 rad) CD45.1/2 recipients. Mixed chimaeras were
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LETTER RESEARCH analysed 5–7 weeks post-reconstitution. Populations of interest were normalized to the reconstitution ratio of each chimaera, as determined by the CD45.21/ CD45.11 ratio of BM lymphocytes or splenic B cells. Quantitative PCR with reverse transcription. RNA was isolated from 5 3 103– 10 3 103 PLZFhigh, CLP, iILC2, NK and ILC3-enriched LPL using the Arcturus PicoPure RNA Isolation kit (Applied Biosystems) and reverse transcribed using the USB First-Strand cDNA Synthesis kit (Affymetrix). The following TaqMan primers (Applied Biosystems) were used (Probe ID in parenthesis): Gata3 (Mm00484683_ m1), Hprt1 (Mm00446968_m1), Id2 (Mm00711781_m1), Rora (Mm00443103_m1),
Rorc (Mm01261022_m1), Tbx21 (Mm00450960_m1), Tcf7 (Mm00493445_m1), Tox (Mm00455231_m1) and Zbtb16 (Mm01176868_m1). Quantitative PCR with reverse transcription was performed on an Mx3005p machine (Stratagene). Statistical analysis. Two-tailed Student’s t-test was performed using Prism (GraphPad Software) to determine whether data differed from the expected value. *P , 0.05; **P , 0.001; ***P , 0.0001. 29.
Lee, E. C. et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73, 56–65 (2001).
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Extended Data Figure 1 | PLZF expression and lineage tracing in PLZFGFPcre mice. a, A sequence encoding an IRES and a GFP-cre fusion protein was inserted immediately after the Zbtb16 stop codon in C57BL/6J ES cells and knock-in mice were bred to ACTB-FLPe mice to excise the neomycin resistance cassette and generate the PLZFGFPcre allele. b, FACS analysis of the indicated populations from PLZFGFPcre1/2 ROSA26-YFP mice. c, Summary of data (mean 6 s.e.m.) from 2–5 mice analysed in 2 or more independent experiments.
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Extended Data Figure 2 | Gating strategy for analysis of ILC and LTi among LPL. ILC2 cells were identified as IL-7Ra1KLRG11 among CD3e2CD192 LPL (top left), and then gated Thy1.21 (not shown). CD3e2CD192 LPL were gated as IL-7Ra1KLRG12 (top left) and then subsetted into CCR61CD41 (CD41 LTi cells) and CCR61CD42 (CD42 LTi cells) (bottom left). NCR1 ILC3 were identified as CD3e2CD192 LPL that expressed NKp46 but not NK1.1 (top right).
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Extended Data Figure 3 | Transcription factor expression by PLZFhigh bone marrow precursors. Quantitative PCR with reverse transcription analysis for Tbx21 and Rora as indicated. NKP are Lin2CD271IL-7Ra2Flt32CD1221 BM cells. Mean 6 s.e.m. of data from 2–3 independent experiments.
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Extended Data Figure 4 | PLZFhigh-derived NK1.11 cells are distinct from CLP-derived NK1.11 cells. CD45.2 Rag22/2Il2rg2/2 mice were injected with equivalent numbers of CD45.2 PLZFhigh cells and CD45.1 CLP (800 of each) and the resulting NK1.11CD3e2TCRb2 cells present in the spleen were analysed 5–7 weeks later by FACS, as indicated. Note that PLZFhigh-derived cells expressed higher amount of surface NKp46, whether they were identified as CD45.21 or as CD45.12 in reciprocal staining experiments. Similar results were obtained for lung NK1.11 cells. Data representative of 5 chimaeras from 2 independent experiments.
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Extended Data Figure 5 | FTOC of PLZFhigh cells. FACS analysis of PLZFhigh and CLP cells (100 of each) co-cultured for 15 days in FTOC (a). The percentages of PLZFhigh- or CLP-derived cells that are CD3e1 are summarized in the bar graph (b). Data representative of 7 independent cultures.
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Extended Data Figure 6 | Additional characterization of PLZFhigh cells after culture on OP9 cells. a, FACS analysis of PLZFhigh or CLP cells from adult BM cultured on OP9 for 4 days showing expression of T1/ST2 on ICOShigh cells. Data representative of 4 replicate cultures from 2 independent experiments. b, FACS analysis of fetal liver PLZFhigh cells after culture on OP9 for 7 days, showing expression of GATA3 by ICOShigh cells and RORct by ICOSint cells. Data representative of 2 independent experiments.
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Extended Data Figure 7 | Proposed model of ILC development. A CLP-derived IL-7Ra1a4b71 population bifurcates into RORcthigh LTi precursors (LTiP) and PLZFhigh ILCP, the latter of which gives rise to all ILC lineages. Whether NKP cells develop directly from CLPs or progress through an IL-7Ra1a4b71 stage has yet to be determined.
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